Central to the form and function of the heart are the myofibrils that provide the cytoskeletal structure and the contractile force that characterize cardiomyocytes. The first strategy of the experimental approaches is on analyzing myofibril formation inside living cardiomyocytes via the transfections of plasmids encoding sarcomeric proteins linked to Green or Blue Fluorescent Proteins, the microinjection of peptide fragments and fluorescently labeled cytoskeletal proteins coupled to the use of quantitative optical techniques. The second approach is focused on a molecular analysis of interacting proteins in the Z-bodies and Z-bands of the assembling myofibrils. The first specific aim is to analyze the assembly of sarcomeric subunits into myofibrils in cultures of living cardiomyocytes under normal spreading conditions, after cytokinesis and during hypertrophy. The second specific aim is to determine if the formation of premyofibrils and their transformation to mature myofibrils in living cardiomyocytes requires non-muscle myosin II whose function is activated through the Rho family of G-Proteins. The third specific aim is to analyze the role of alpha-actinin in the formation of the Z-bodies in premyofibrils and the recruitment and role of vinculin in the fusion of the Z-bodies to form the costameric Z-bands of mature myofibrils in living cardiomyocytes. The fourth specific aim is to use a GFP targeting assay to determine which subdomains of the N-terminus of titin can localize to the Z-bands of living cardiac muscle cells. We will probe the role of these different titin domains in targeting costameric proteins to the Z-band. Protein-protein interaction assays will be used to identify the Z-band and costameric proteins, that interact with the Z-band regions of titin. The advanced optical methods coupled with molecular biological techniques will allow hypotheses about the formation of myofibrils to be tested directly in single living rat neonatal and chick embryonic cardiomyocytes. These approaches should yield new insights into basic and pathologic processes in myofibril assembly in living cardiomyocytes.